The objective of this study is to elucidate the intracellular interactions of the Human Immunodeficiency Virus (HIV) regulatory protein, Nef, with host cell activities. Early in the HIV-1 infective process this retrovirus expresses regulatory proteins, with the Nef transcript representing nearly 80% of total viral mRNA. Recent work by Desrosiers and colleagues has shown that in vivo infectivity by the closely related simian form of HIV is achieved in the absence of Nef expression, but that there is an absolute requirement for Nef in the development of the immunodeficiency. This essential activity of the Nef protein in the development of AIDS has not been defined. Nef expression in T cell lines has been achieved by transduction with a retroviral expression vector. Nef expression was found to down-modulate the surface expression of CD4 and CD8a-beta, the two molecules known to interact with the T cell specific tyrosine kinase, p56(lck). Additionally, we have found that Nef expression in a murine T cell hybridoma sensitizes that cell to T cell receptor stimulation; that is, Nef lowers the biochemical threshold for T cell activation. To elucidate this Nef activity we have developed a murine T cell hybridoma line in which activation, as defined by the T cell specific synthesis of IL2, is dependent upon Nef expression. With this cell line we have found that Nef functions as or through a tyrosine kinase.